This invention is directed to a method of synthesizing libraries of diverse and complex highly substituted 2,4-dioxopiperidine and novel intermediate compounds. More particularly, the invention relates to a method of solid phase synthesis of (E)-N-substituted-acetyl-N-(2-methoxycarbonyl-3-(Aryl)-prop-2-enyl)amino acids and condensation of the intermediate esters using commercially available Fmoc-protected amino acids on Wang Resins. The invention is further directed to methods for synthesizing the libraries on solid supports. The invention is also directed to methods for identifying and isolating the highly substituted 2,4-dioxopiperidine and novel intermediate compounds with useful and diverse activities from such libraries.
Compounds having biological activity can be identified by screening diverse collections of compounds (i.e., libraries of compounds) produced through molecular biology techniques or synthetic chemical techniques.
The generation of chemical libraries on and off solid resins have proven to be a valuable resource for the pharmaceutical industry in their endeavors to discover new drugs using high throughput screening (HTPS) techniques. In creating the libraries, the compounds are ideally synthesized in situ in solution phase or on a solid support. However, relatively simple synthetic methods to produce a diverse collection of such derivatives in situ are often not available.
Such screening methods include methods wherein each member of the library is tagged with a unique identifier tag to facilitate identification of compounds having biological activity or where the library comprises a plurality of compounds synthesized at specific locations on the surface of a solid substrate wherein a receptor is appropriately labeled to identify binding to the compound, e.g., fluorescent or radioactive labels. Correlation of the labeled receptor bound to the substrate with its location on the substrate identifies the binding compound. Using these techniques, the development of efficient high throughput screening has greatly enhanced the pharmaceutical industry""s ability to screen large numbers of compounds for biological activity.
Central to these methods is the screening of a multiplicity of compounds in the library and the ability to identify the structures of the compounds that have a requisite biological activity. Preferably, in order to facilitate synthesis and identification, the compounds in the library are typically formed on solid supports wherein the compound is covalently attached to the support via a cleavable or non-cleavable linking arm. In this regard, libraries of diverse compounds are prepared and then screened to identify xe2x80x9clead compoundsxe2x80x9d having good binding affinity to the receptor.
Pharmaceutical drug discovery relies heavily on studies of structure-activity relationships wherein the structure of xe2x80x9clead compoundsxe2x80x9d is typically altered to determine the effect of such alteration on activity. Alteration of the structure of the lead compounds permits evaluation of the effect of the structural alteration on activity.
Thus, libraries of compounds derived from a lead compound can be created by including derivatives of the lead compound and repeating the screening procedures. In this manner, compounds with the best biological profile, i.e., those that are most active and which have the most ideal pharmacologic and pharmacokinetic properties, can be identified from the initial lead compound.
Recently various 2,4-Dioxopiperidines were found to be potent as therapeutic or prophylactic agents for hepatic disease, for bacterial and viral infections and for diseases, including AIDS. Eur. Pat. No. 149534; WO 95/14012.
Since 2,4-dioxopiperidines have been shown to possess diverse pharmacological and chemical properties and biological activity in a number of therapeutic areas, the generation of a 2,4-dioxopiperidine compound library would be useful as a screening tool.
Accordingly, in order to develop new pharmaceutical drugs to treat various disease conditions, it would be highly desirable to be able to generate such libraries of substituted 2,4-dioxopiperidine and novel intermediate compounds optionally attached to a solid support. Thus, there is a need for a facile in situ method for the generation of a multiplicity of highly substituted 2,4-dioxopiperidine and novel intermediate compounds.
The present invention is directed to a process that allows for the assembly of diverse, highly substituted 2,4-dioxopiperidine and novel intermediate compounds using commercially available Fmoc-protected amino acids on solid resins. The rapid synthesis of such highly complex drug like molecules with or without defined stereochemistry is unexpected and surprising.
Accordingly, the invention is directed to a method of synthesizing highly substituted 2,4-dioxopiperidine compounds having the general Formula 1: 
wherein:
R1 is selected from the group consisting of a standard natural amino acid side chain, CH3, CH(CH3)CH2CH3 and CH2 Phenyl;
R2 is selected from the group consisting of alkyl, substituted alkyl, aralkyl, phenyl and substituted aryl;
R3 is selected from the group consisting of COOalkyl, CN, COloweralkyl, COaralkyl (benzyl and substituted benzyl) and COaryl; where aryl is phenyl, substituted phenyl, thienyl, furyl and napthyl; which method comprises,
(a) preparing a compound of the formula: 
wherein R1 to R3 are as described above, on a solid resin support;
(b) cleaving the compound from the solid resin support and methylating to yield an intermediate methyl ester compound of the formula: 
and,
(c) condensing the intermediate methyl ester compound to provide the substituted 2,4-dioxopiperidine compounds of Formula 1.
This invention is also directed to highly substituted novel intermediate compounds assembled by the methods of the present invention having the general formula: 
wherein R1 to R3 are as recited above.
In one embodiment of this invention, the amino acid derived novel intermediate precursors are covalently attached to a solid support. Solid supports containing such amino acid derived intermediate precursors may comprise a linking arm which links the solid support to the intermediate precursor. The linking arm can be either cleavable or non-cleavable. The novel intermediate precursors attached to the solid support can be used to prepare a library of solid phase 2,4-dioxopiperidine derivatives.
The methods described above can be used to create a library of diverse dioxopiperidine derivatives. Accordingly, in one of its composition aspects, this invention is directed to a library of diverse (E)-N-substituted-acetyl-N-(2-methoxycarbonyl-3-(Aryl)-prop-2-enyl)amino acid derivatives comprising a plurality of solid supports having a plurality of covalently bound (E)-N-substituted-acetyl-N-(2-methoxycarbonyl-3-(Aryl)-prop-2-enyl)amino acid derivatives, wherein the derivatives bound to each of said supports is substantially homogeneous and further wherein the derivatives bound on one support is different from the derivatives bound on the other supports. The library is screened to isolate individual compounds that bind to a receptor or possess some desired property.
Relative to the above generic description, certain compounds of the general formulae are preferred.
Particularly preferred embodiments are those compounds in which:
R1 is selected from the group consisting of a standard natural amino acid side chain, CH3, CH(CH3)CH2CH3 and CH2 Phenyl;
R2 is selected from the group consisting of alkyl, aralkyl, phenyl and substituted phenyl; where the substituents on the substituted phenyl group are selected from lower alkyl, halo, alkoxy, nitrile, cyano, alkoxycarbonyl, aryloxycarbonyl, nitro, furyl and substituted furyl; where the substituents on the substituted furyl group are selected from alkyl, halo, alkoxy, alkoxycarbonyl, nitrile, aryloxycarbonyl, nitro, thienyl and substituted thienyl; where the substituents on the substituted thienyl group are selected from lower alkyl, halo, alkoxy, nitrile, alkoxycarbonyl, aryloxycarbonyl, naphthyl and substituted naphthyl; where the substituents on the substituted naphthyl group are selected from lower alkyl, halo, alkoxy, nitrile, alkoxycarbonyl and aryloxycarbonyl; and,
R3 is selected from the group consisting of COOalkyl, CN, COloweralkyl, COaralkyl (benzyl and substituted benzyl) and COaryl; where aryl is phenyl, substituted phenyl, thienyl, furyl and napthyl.
Preferred embodiments are those compounds wherein:
R1 is selected from the group consisting of CH3, CH(CH3)CH2CH3 and CH2 Ph;
R2 is selected from the group consisting of phenyl, halophenyl, cyanophenyl and carboxyphenyl; and,
R3 is selected from the group consisting of COOC2H5, CN and COCH3.
Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group.
The standard amino acid side chain for R1 may be selected from any of the known natural amino acid groups such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, isoleucine, glycine, leucine, histidine, methionine, lysine, phenylalanine, proline, serine, valine, threonine, tryptophane, tyrosine, and the like.
The term xe2x80x9calkylxe2x80x9d refers to straight or branched chain unsubstituted hydrocarbon groups of 1 to 20 carbon atoms, preferably 1 to 7 carbon atoms. The expression xe2x80x9clower alkylxe2x80x9d refers to unsubstituted alkyl groups of 1 to 4 carbon atoms.
The term xe2x80x9csubstituted alkylxe2x80x9d refers to an alkyl group substituted by, for example, one to four substituents, such as, halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, cycloalkyl, cycloalkyoxy, heterocylooxy, oxo, alkanoyl, aryloxy, alkanoyloxy, amino, alkylamino, arylamino, aralkylamino, cycloalkylamino, heterocycloamino, disubstituted amines in which the 2 amino substituents are selected from alkyl, aryl or aralkyl, alkanoylamine, aroylamino, aralkanoylamino, substituted alkanoylamino, substituted arylamino, substituted aralkanoylamino, thiol, alkylthio, arylthio, aralkylthio, cycloalkylthio, heterocyclothio, alkylthiono, arylthiono, aralkylthiono, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, sulfonamido (e.g. SO2NH2), substituted sulfonamido, nitro, cyano, carboxy, carbamyl (e.g. CONH2), substituted carbamyl (e.g.CONH alkyl, CONH aryl, CONH aralkyl or cases where there are two substituents on the nitrogen selected from alkyl, aryl or aralkyl), alkoxycarbonyl, aryl, substituted aryl, guanidino and heterocyclos, such as indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl and the like. Where noted above that the substituent is further substituted, such substitutions will be with halogen, alkyl, alkoxy, aryl or aralkyl.
The term xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d refers to fluorine, chlorine, bromine and iodine.
The term xe2x80x9carylxe2x80x9d refers to monocyclic or bicyclic aromatic hydrocarbon groups having 6 to 12 carbon atoms in the ring portion, such as phenyl, naphthyl, biphenyl and diphenyl and diphenyl groups, each of which may be substituted.
The term xe2x80x9caralkylxe2x80x9d refers to an aryl group bonded directly through an alkyl group, such as benzyl.
The term xe2x80x9csubstituted arylxe2x80x9d refers to an aryl group substituted by, for example, one to four substituents such as alkyl, substituted alkyl, halo, trifluoromethoxy, trifluoromethyl, hydroxy, alkoxy, cycloalkyl, cycloalkyloxy, heterocyclooxy, alkanoyl, alkanoyloxy, amino, alkylamino, aralkylamino, cycloalkylamino, heterocycloamino, dialkylamino, alkanoylamino, thiol, alkylthio, cycloalkylthio, heterocyclothio, ureido, nitro, nitrile, cyano, carboxy, carboxyalkyl, carbamyl, alkoxycarbonyl, alkylthiono, arylthiono, alkysulfonyl, sulfonamido, aryloxy, alkoxycarbonyl, nitrile, furyl and the like. The substitutent may be further substituted by halo, hydroxy, alkyl, alkoxy, alkoxycarbonyl, nitrile, aryl, aryloxycarbonyl, substituted aryl, substituted alkyl, aralkyl, heterocyclyl and substituted heterocyclyl. xe2x80x9cSubstituted benzylxe2x80x9d refers to a benzyl group substituted by, for example, any of the groups listed above for substituted aryl.
The term xe2x80x9ccycloalkylxe2x80x9d refers to optionally substituted, saturated cyclic hydrocarbon ring systems, preferably containing 1 to 3 rings and 3 to 7 carbons per ring which may be further fused with an unsaturated C3 to C7 carbocyclic ring. Exemplary groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, and adamantyl. Exemplary substituents include one or more alkyl groups as described above or one or more groups described above as alkyl substituents.
The terms xe2x80x9cheterocyclexe2x80x9d, xe2x80x9cheterocyclicxe2x80x9d and xe2x80x9cheterocycloxe2x80x9d refer to an optionally substituted, fully saturated or unsaturated, aromatic or nonaromatic cyclic group. Such group, for example, can be a 4 to 7 membered monocyclic, a 7 to 11 membered bicyclic or a 10 to 15 membered tricyclic ring system which has at least one heteroatom in at least one carbon atom containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1, 2 or 3 heteroatoms selected from nitrogen atoms, oxygen atoms and sulfur atoms, where the nitrogen and sulfur heteroatoms may also optionally be oxidized and where the nitrogen heteroatoms may also optionally be quaternized. The heterocyclic group may be attached at any heteroatom or carbon atom.
Exemplary monocyclic heterocyclic groups can include pyrrolidinyl, pyrrolyl, indolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thizaolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxazepinyl, azepinyl, 4-piperidonyl, pyridyl, N-oxo-pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, tetrahydropryanyl, tetrahydrothiopyranyl, tetrahydrothiopyranyl sulfone, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dixolane and tetrahydro-1,1-dioxothienyl, dioxanyl, isothiazolidinyl, thietanyl, thiiranyl, triazinyl, triazolyl and the like.
Exemplary bicyclic heterocyclic groups include benzothiazolyl, benzoxazolyl, benzothienyl, quinuclidinyl, quinolinyl, quinolinyl-N-oxide, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopridyl, furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,1-b]pyridinyl), or furo[2,3-b]pyridinyl), dihydroisoindolyl, dihydroquinazolinyl (such as 3,4-dihydro-4-oxo-quinazolinyl), benzisothiazolyl, benzisoxazolyl, benzodiazinyl, benzofurazanyl, benzothiopyranyl, benzotriazolyl, benzpyrazolyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, dihydrobenzopyranyl, indolinyl, isochromanyl, isoindolinyl, naphthyridinyl, phthalazinyl, piperonyl, purinyl, pyridopyridyl, quinazolinyl, tetrahydroquinolinyl, thienofuryl, thienopyridyl, thienothienyl and the like.
Exemplary substituents include one or more alkyl groups as described above or one or more groups described above as alkyl substituents.
The term xe2x80x9cheteroatomsxe2x80x9d shall include oxygen, sulfur and nitrogen.
Throughout this specification, certain abbreviations are employed which have the following meanings, unless specifically indicated otherwise: DMF is N,N-dimethyl formamide, MeOH is methanol, THF is tetrahydrofuran, DME is ethylene glycol dimethyl ether, NBS is N-bromosuccinimide, Fmoc is N-(9-fluorenylmethoxycarbonyl), NOE is Nuclear Overhauser Enhancement, DMS is dimethyl sulfide, DMAP is dimethylaminopyridine, DABCO is 1,4-diazabicyclo[2.2.2]octane, DIC is diisopropylcarbodimide, TFA is trifluoroacetic acid and TMSCHN2 is trimethylsilyldiazomethane.
Representative compounds of the present invention are synthesized in accordance with the general method described below and illustrated in Scheme 1. Since Scheme 1 is an illustration, the invention should not be construed as being limited by the chemical reactions and conditions expressed.
Various publications are cited throughout the description for this general scheme. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains. 
This invention is directed to the solid phase synthesis of diverse, highly substituted 2,4-dioxopiperidine Compounds 1 g from intermediate (E)-N-substituted-acetyl-N-(2-methoxycarbonyl-3-(Aryl)-pro p-2-enyl)amino acid Compounds 1f using commercially available Fmoc-protected amino acid Compounds 1a on Wang Resins. The synthesis requires an alkylation and subsequent amidation step The use of the Fmoc-protected amino acids on Wang Resins and the alkylation and amidation steps provides unique and novel diversity to the highly substituted 2,4-dioxopiperidine Compounds 1g and intermediate Compounds 1f.
This approach can be used to generate a number of compound libraries of both the highly substituted 2,4-dioxopiperidine Compounds 1g and the highly substituted intermediate Compounds 1f with the three diversity elements R1, R2 and R3. By condensing the highly substituted intermediate Compounds If, the intermediate esters can be further elaborated to the various highly substituted 2,4-dioxopiperidine Compounds 1g.
The solid phase synthesis was initiated with the deprotection of the Fmoc-protected amino acid Compound 1a on Wang Resins (1, 2). Piperidine in DMF was added and the reaction was run for about 2 h at about room temperature. Various commercially available Fmoc-amino acid-Wang Resin units provide the subsequent diversity element in R1 of the intermediate Compounds 1f and 2,4-dioxopiperidine Compounds 1g.
After removal of the Fmoc group from Compound 1a, alkylation of the free amine Compounds 1b was accomplished by a palladium (0) catalyzed reaction with methyl 2-(Z)-(bromomethyl)-3-aryl-prop-2-enoate Compounds 1c in DMF run for about 16 h at about room temperature.
The bromide Compounds 1c were synthesized in two steps. The first step is a DABCO catalyzed Baylis-Hillman""s reaction of methyl acrylate with the appropriate aldehyde (3, 4), run for about 6 h at about 0xc2x0 C. and then stirred at about room temperature for about 16 h. The second step involves a Corey-Kim bromination using NBS-DMS (5, 6) in CH2Cl2 cooled to about 0xc2x0 C. and stirred for about 1 h. The solution of Compound 1b was added and stirred for diversity element in R2 of the intermediate Compounds 1f and 2,4-dioxopiperidine Compounds 1g.
The subsequent amidation step generates the third diversity element in R3 of the intermediate Compounds if and 2,4-dioxopiperidine Compounds 1g. By using different amidating reagents and conditions, such as stirring with ethylmalonyl chloride in DMAP and CH2Cl2 for about 16 h at about 25xc2x0 C. or stirring with cyanoacetic acid and DIC in DMF for about 50 h at about 25xc2x0 C. or stirring with diketene for about 50 h at about 25xc2x0 C., the compounds, respectively, xcex1-carboethoxyamide (7), xcex1-cyanoamide (8) and xcex1-ketoamide (9) can be produced.
After resin cleavage with CF3COOH/MeOH by stirring for about 2 h at about 25xc2x0 C. and methylation with TMSCHN2 in MeOH, the intermediate methyl ester Compounds 1f were obtained. The total yield for the reaction sequence from Compounds 1a to Compounds 1f is from about 28% to about 31%.
Dieckmann condensation of intermediate Compounds 1f with t-BuOK in THF stirred for about 3 h at about xe2x88x9278xc2x0 C. provided 2,4-dioxopiperidine Compounds 1 g in yields from about 60% to about 72% (10, 11).
Compounds 1f and Compounds 1g were characterized by 1HNMR, 13C NMR and ESMS. The (E)/(Z) configurations were determined by NOE experiments.
The synthesis of specific, representative compounds of the present invention is presented in detail in the following examples. These examples are intended to illustrate the methods of synthesis and are not intended to limit the scope of the claims in any way. Moreover, no attempt has been made to optimize the yields obtained in these reactions. It would be obvious to one skilled in the art that variations in reaction times, temperatures, solvents, and/or reagents could increase the yields.